Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsis-like illness with cytopenia, hepatosplenomegaly and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We have shown that freshly isolated, resting natural killer (NK) cells and CD8+ T cells express syntaxin-11. In infants, NK cells are the predominant perforincontaining cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Interestingly, IL-2stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared to FHL2 and FHL3 patients. Since the effector T cell compartment is still immature in infants, our data suggest that the observed defect in NK cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.[unreadable] [unreadable] The lymphokine interleukin (IL)-15 is essential for the development, survival, and proliferation of NK cells. A peculiar feature of stimulation by IL-15 is that expression of the alpha chain of the IL-15 receptor (IL-15Ralpha) is not required on NK cells but on other cells, such as dendritic cells, that trans-present IL-15 to the low affinity IL-15 receptor (IL-15R beta-gamma) on NK cells. Trans-presentation of IL-15 in the context of a cell-to-cell contact raises the possibility of regulation by other receptorligand interactions between the responding NK cell and the presenting cell. Does proper stimulation of NK cells by IL-15 bound to the IL-15Ralpha require additional signals from other receptors? Likewise, is IL-15 trans-presentation regulated by NK cell inhibitory receptors? These are important questions given the essential role of IL-15 in several facets of NK cell biology. To address the first question, the IL-15Ralpha chain has been expressed on the Drosophila insect cell line S2. The use of a cell from an evolutionary distant organism, such as the fruit fly Drosophila, has the advantage of reducing the complexity due to the many receptorligand interactions between human NK cells and mammalian target cells. Specific ligands of individual NK cell receptors can be expressed on S2 cells and tested for their ability to induce signals and functional responses in NK cells. Expression of IL-15Ralpha on insect S2 cells was sufficient to bind IL-15 and to trans-present it to NK cells. Co-expression of ICAM-1, a ligand for the adhesion receptor LFA-1, with the IL-15Ralpha did not result in greater proliferation than with the IL-15Ralpha alone. Therefore, stimulation of NK cells by trans-presented IL-15 does not require binding of other NK cell receptors to human ligands on the presenting cell. Furthermore, co-engagement of the IL-15R beta-gamma with an inhibitory receptor, through expression of MHC class I on Drosophila S2 cells, caused a reduction in the proliferation of resting NK cells. Regulation of IL-15 trans-presentation by MHC class I-specific inhibitory receptors could be an important mechanism for the termination of NK cell responses. As inflammatory conditions induce greater cell surface expression of MHC class I, one may expect down-regulation of IL-15 trans-presentation in the later phase of NK cell immune responses. [unreadable] [unreadable] Most receptors in the immune system are expressed at the surface of immune cells where they can sense the environment and induce appropriate responses, such as attraction to chemotactic signals, adhesion to other cells, and even attack of other cells that have been marked for elimination. However, a few receptors reside transiently in endosomes (e.g. growth factor receptors, toll-like receptors) from where they can transmit signals to the cell. Signaling from endosomes has emerged as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. A receptor of the killer cell Ig-like receptor (KIR) family, called KIR2DL4, has the unique property of residing almost exclusively in endosomes. Unlike other KIRs that mediate inhibition of NK cell cytotoxicity, KIR2DL4 triggers the production of a limited set of cytokines and chemokines, and a few other molecules. The profile of genes activated by KIR2DL4 carries a typical pro-inflammatory and pro-angiogenic signature. The biological context in which KIR2DL4 may provide a useful response is early pregnancy, where the KIR2DL4 ligand HLA-G is produced. The non-classical major histocompatibility class I molecule HLA-G is expressed by trophoblast cells from the fetus that invade the maternal uterus. Uterine tissue in early human pregnancy is characterized by extensive remodeling of the vasculature, invasion of fetal trophoblast cells, and by an abundance of maternal NK cells. How NKtrophoblast cell interactions influence vascular remodeling is unknown. Two major questions raised by these findings are the mechanism for transport of KIR2DL4 to endosomes, and the signaling pathway triggered by KIR2DL4 once it is bound to a soluble ligand within endosomes. By use of tandem affinity purification and mass spectrometry, we have identified a new signaling molecule associated with KIR2DL4. We are in the process of identifying the critical steps in this pro-inflammatory signaling pathway.